Understanding the capacity of the cellular immune response to clear Borrelia burgdorferi, the etiologic agent of Lyme disease, is essential to a proper understanding of the pathogenesis of Lyme disease. We have shown previously that macrophages take up and kill B. burgdorferi rapidly in vitro. Despite this killing, we have observed occasional cell-associated persistent spirochetes and such spirochetes have been recultured. Ex vivo, we have isolated spirochetes from the uninflammed peritoneal cavity of chronically-infected mice despite the presence of resident macrophages. We will examine this failure of normal killing mechanisms in vivo in three ways: in vitro, ex vivo, and in situ. Our methodologies include biochemical assays and quantitative confocal fluorescent and electron microscopy. Specifically, in Aim 1 we will identify kinetics, extent, and mechanisms of killing of B. burgdorferi by phagocytes in vitro as a necessary first step to Aim 2: testing the killing capacity of peritoneal macrophages isolated from B. burgdorferi-infected mice, in the presence and absence of lavage fluid which may contain immunomodulatory elements. In case characteristics of the persistent spirochetes are responsible for their faulty clearance, we will also examine uncultured spirochetes isolated from infected animals, by immunofluorescent and electron microscopic labeling of expressed surface proteins. Finally, in Aim 3, we will study these questions in situ using the cardiac involvement of Lyme borreliosis, an ideal disease presentation for these purposes, as the lesion in question appears to be primarily macrophage-mediated. We will examine sections of cardiac and other tissue from infected mice and evaluate whether the macrophages are phagocytosing and degrading spirochetes in the heart; whether the macrophages are activated or deactivated as determined by a panel of marker antibodies; and finally, whether modifying the state of activation of the macrophages with cytokines can influence the progression of Lyme carditis, a prospect with obvious therapeutic implications. Taken as a whole, we expect these studies to provide a comprehensive picture of the functional integrity of phagocytic cells in a host infected with B. burgdorferi.